Most of the fermentation experiment designs were limited by the low-throughput of shake flask, especially for the medium optimization. A simple high-throughput screening system was developed for the determination of pigment in Monascus purpureus fermentation samples. This downscaled system was designed to optimize medium composition combined with statistical methods. The total 29 experiments designed by the Box-Behnken were used to study the 4 most important operating variables on pigment production. The analysis revealed that the optimum concentrations of glucose, peptone, $NaNO_3$, and $KH_2PO_4$ were 51.42, 4.91, 1.00, and 1.00 g/L, respectively. A production of 69.5 U/mL was achieved in agreement with the prediction (68.9 U/mL) fermented in 24-deep-well microtiterplates. Furthermore, the fermentation medium optimized in the high-throughput system was verified in shake flasks, and the pigment production could be enhanced from 206.5 U/mL in un-optimized medium to 265.8 U/mL, giving nearly 1.30-fold increase in production.